Project Summary The long-term goal of this project is to understand how mRNA translational regulatory mechanisms control cellular stress response under physiological and pathological conditions. The specific pathway of interest is the Integrated Stress Response (ISR), which involves stress-responsive kinases that phosphorylate the ? subunit of eIF2 to attenuate general translational initiation. More recently, we discovered that ISR inhibits mRNA translation through the induction of yet another translational inhibitor 4E-BP. At the same time, such conditions paradoxically stimulate the translation of transcription factors such as ATF4, due to the presence of regulatory upstream Open Reading Frames (uORFs) that precede the main ATF4 ORF. How stress responsive transcripts evade translational inhibition, or even undergo more active expression, in stressed cells is one of the major conceptual questions that remains poorly understood. Our preliminary studies using Drosophila have led us to a number of new insights to this question: These include our findings that (1) ISR boosts innate immune response to bacterial infection and anti-microbial peptides have 5?UTRs that can evade translational inhibition imposed by ISR signaling, (2) that previously unexpected factors regulate the activation of ISR, and (3) that ATF6 also has a 5?UTR that stimulates the main ORF translation in response to stress. Here I propose to use Drosophila to investigate the underlying regulatory mechanisms and determine how the newly identified ISR regulatory factors affect innate immune response, inflammation, and lifespan of Drosophila. We will supplement the molecular genetics-based approach with genomic tools such as ribosome profiling and structure-based modeling studies. Three Specific Aims will be pursued: (1) We will determine how ISR signaling enhances innate immune response, mainly focusing on the idea that anti-microbial peptide transcripts undergo mRNA translation through an unconventional mechanism to evade translational inhibition associated with ISR signaling. (2) Through the characterization of the novel ISR regulators that we have identified, we plan to determine how uORF containing transcripts increase their translation when general mRNA translation is suppressed, and examine how they affect phenotypes associated with abnormal ISR regulation. (3) We will test the hypothesis that ATF6 is another transcription factor that is regulated at the level of mRNA translation during ISR to mediate its transcriptional response. Notably, impairment or excessive stimulation of this pathway underlies various neurodegenerative and metabolic disorders in humans. Therefore, a better understanding of the regulatory mechanisms will not only advance our conceptual understanding of gene expression regulation in cells under stress but also may prompt the development of new strategies to modulate ISR signaling for therapeutic purposes.